Co-authored by Dr. Jeffrey Rohrer, Director of Applications Development, Dionex Products for Thermo Fisher Scientific.
Glycosylation is an important post-translational modification that plays a key role in normal cell function. It is implicated in many disease processes, including those associated with cancer, arthritis and diabetes. One of the most diverse forms of protein glycosylation is O-glycosylation, which involves glycan attachment via the oxygen on serine or threonine amino acid residues.
Studying O-glycosylation is demanding due to the large variability in O-glycan composition and linkage, which complicates structural analysis. This challenge is further compounded by the lack of a universal enzyme for O-glycan release. To reveal insight into O-glycosylation processes and their implications for human health, reliable analytical methods capable of high-resolution chromatographic separation and accurate identification of glycans are essential.
O-linked glycan profiling using HPAE-PAD/MS
High-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD) and mass spectrometry (MS) is a well-established and powerful technique for glycan analysis. HPAE is capable of highly selective glycan separations using strong anion-exchange column chemistries, while PAD and MS enable direct quantification of analytes, eliminating the need for time-consuming sample derivatization steps.
Recent years have seen substantial improvements in the capabilities of HPAE-PAD/MS component technologies. Notably, advances in the separation resolution offered by modern HPAE columns, as well as the accuracy of high-resolution accurate mass (HRAM) techniques, have resulted in substantial improvements in the quality of structural information that can be obtained from O-glycan analyses.
Putting advanced HPAE-PAD/MS techniques for O-glycan profiling to the test
To demonstrate the potential of modern HPAE-PAD/MS systems for O-glycan profiling, we characterized the glycans liberated from four glycoproteins (porcine gastric mucin type III, bovine fetuin, bovine fibrinogen, and bovine thyroglobulin), as reported in this application note.
O-linked glycans were released from the glycoproteins under alkaline conditions by reductive β-elimination. To prevent glycan degradation, the reducing ends of the carbohydrates were converted to base-stable alditols. Following purification with porous graphitized carbon resin, the samples were analyzed using a Thermo Scientific™ Dionex™ ICS-5000+ Ion Chromatography System (equivalent to ICS-6000) employing a Thermo Scientific™ Dionex™ CarboPac™ PA300-4μm column. A sodium hydroxide and sodium acetate-based eluent gradient was used for the separation, and the column effluent was passed through a Dionex™ ERD™500 (2 mm) desalter to remove sodium ions prior to MS analysis.
Glycans eluting from the column were detected simultaneously by PAD and MS. A Thermo™ Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer operating in negative electrospray mode was used to reveal detailed insight on glycan linkages. Fragmentation by higher energy collision-induced dissociation provided information-rich MS2 spectra dominated by glycosidic and cross-ring fragments. Structures were confirmed by annotating the diagnostic fragmentation patterns observed in the MS2 spectra.
Driving improvements in high-resolution O-glycan separation and HRAM analysis
Representative PAD and MS base peak chromatograms showing O-linked glycans liberated from the porcine gastric mucin type III glycoprotein sample are shown in Figure 1. The released glycans included a diverse range of structures, varying in size, linkage position, stereochemistry, monosaccharide composition and sulfate groups. The mass accuracy of the detected analytes was less than 5 ppm, ensuring high confidence in peak annotation. Excellent separation of small, neutral glycans (eluting between 4.2 and 30.0 mins), sialylated glycans (33.1 to 35.0 mins), and charged, sulfated glycans (after 35.0 mins) was achieved. Similarly, efficient separation of O- and N-glycans also was achieved for the other three glycoproteins.
Key to the robust performance of this method is the high-resolution separation afforded by the Dionex™ CarboPac™ PA300-4μm column, which permits simultaneous analysis of neutral and charged glycans. Compared with older columns, this column improves the separation of neutral glycans eluting at the beginning of the gradient. No enrichment or derivatization steps are required prior to analysis, preserving the sample’s native glycan profile without losing structural information or adding ambiguous signals through extraneous labeling reactions. This enables rapid and reliable glycan characterization, with a high degree of confidence in structural identification.
Reliable structural insight from O-glycan profiling
Obtaining meaningful structural insight from O-glycan analyses requires robust methods capable of high-resolution separation and accurate identification of released oligosaccharides. Thanks to improvements in HPAE column chemistries and HRAM technologies, modern HPAE-PAD/MS systems are helping scientists understand O-glycosylation in even greater detail.
You can read more about this method for O-glycan profiling in this application note.
Figure 1. Comparison of (A) PAD chromatogram and (B) MS base peak chromatogram of glycans released from porcine gastric mucin type III.